Photoactivation of fluorescence is a powerful technique for the study of biological molecules in living cells. In particular, this method allows the study of dynamic cellular processes by providing a means to make fiducial marks on biopolymers, monitor biomolecule diffusion, trace cell lineage, and differentiate between two or more populations of a biomolecule (e.g., newly synthesized proteins vs. older proteins, or nucleic acids). Examples of photoactivatable fluorophores include caged fluorescein and photoactivatable green fluorescent protein (PA-GFP). More recently, the use of photochromic proteins such as Kaede (Ando et al., Proc. Natl. Acad. Sci. U.S.A., 99:12651-12656 (2002)) have allowed the discrimination between two different populations of a biomolecule by a shift in the fluorescence emission wavelength after photoactivation.
Photoactivation of PA-GFP can be used to temporally mark a protein population, but it is restricted to green fluorescence. The photoconvertable proteins, Kaede and EOS, (Wiedenmann et al., Proc. Natl. Acad. Sci. U.S.A. 101:15905-15910 (2004)) shift their fluorescence from green to red upon irradiation with long-wave UV light. However, Kaede is an obligate tetramer, and EOS monomer fusion constructs do not express at physiological temperatures. Thus, these proteins can not currently be used in a general method to tag proteins of interest and mark their localization in cells. None of these fluorescent proteins efficiently emit light in the near-IR (>650 nm), where autofluorescence is lowest and light penetration through living tissue is highest. In addition, all of these proteins are relatively large in size (27 kD for the Kaede monomer) and can interfere with the proper localization and function of the protein under study.
Photoactivation of small molecule fluorophores has thus far been mostly limited to o-nitrobenzyl (oNB)-based caged derivatives of coumarin, fluorescein, Q-rhodamine, and resorufin. In these dyes, fluorescence in the caged dye is reduced by either i) alkylation of a free phenolic hydroxyl in the parent dye with an oNB derivative (e.g., present in coumarin, fluorescein and resorufin) or, ii) carbamoylation of a secondary aryl amine with an oNB derivative (e.g., in Q-rhodamine). Photocleavage of the oNB moiety restores the structure of the parent dye, resulting in a large increase in fluorescence. Many exemplary fluorophores either lack suitable free phenol or aryl amine functionality, or such modified derivatives are still fluorescent, and thus are not amenable to the chemical modifications required to render them photoactivatable using this approach. For example, longer-wavelength far-red and near-IR rhodamine and oxazine dyes that have tertiary aryl amines are not compatible with this approach.
While caged-fluorescein is the most widely-used photoactivatable fluorophore, it is highly hydrophobic, poorly soluble in water, and labeled protein tends to aggregate. Moreover, its synthesis is difficult and the fluorescein chromophore bleaches very rapidly. Photocaged coumarin is photoactivated at the same wavelength used to visualize coumarin fluorescence, which greatly limits its use. Photocaged Q-rhodamine is difficult to synthesize and uncages slowly, and caged resorufin is chemically unstable in cell extract. Furthermore, both caged fluorescein and caged Q-rhodamine have two photolabile groups each. Thus, restoration of full fluorescence is a stepwise process, as it requires the removal of both groups, and leads to a heterogeneous population of fluorescent molecules.